Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2525-2528, April 1984 Medical Sciences Chromosome 20 deletion in human multiple endocrine neoplasia types 2A and 2B: A double-blind study (medullary thyroid cancer/pheochromocytoma/calcitonin/oncogenes/gene mapping) V. R. BABU*, DANIEL L. VAN DYKEt, AND CHARLES E. JACKSON Medical Genetics and Birth Defects Center and Division of Clinical Genetics, Department of Medicine, Henry Ford Hospital, Detroit, MI 48202 Communicated by James V. Neel, January 3, 1984 ABSTRACT Multiple endocrine neoplasia type 2A and 2B non-MEN-2 control subjects. All of the MEN families and (MEN-2A and MEN-2B) are autosomal dominantly inherited controls were of European ancestry except for one Black syndromes in which medullary thyroid cancers are associated MEN-2A family. All MEN-2 patients had normal intelli- with adrenal pheochromocytomas. A double-blind analysis of gence and an otherwise normal phenotype. Males and fe- high-resolution G-banded chromosomes was performed on males were about equally represented among the MEN-2 blood specimens from patients in four MEN-2A families and and control subjects. In one of the four MEN-2A families, five MEN-2B (mucosal neuroma phenotype) families and from only medullary thyroid cancers and parathyroid tumors have control subjects. Excluding studies on duplicate blood speci- been detected; in another family, only medullary thyroid mens, 9 of 11 control subjects were scored as having normal cancers and pheochromocytomas have been detected; and in chromosomes 20, and 11 of 14 MEN-2 patients were scored as two families, all three tumor types have been detected. having chromosomal deletion: del(20)(p12.2p12.2) (X2 = 9.00; Three of the MEN-2B (medullary thyroid cancers, mucosal P < 0.001). Two new mutant MEN-2B patients had apparently neuromas, and pheochromocytomas) subjects are members normal chromosomes 20. These findings demonstrate that the of two families, and three are probably new mutants be- dominant mutation in most MEN-2A and MEN-2B families is cause, except for two children of one of them, no relatives a visible chromosome deletion within band 20pl2.2. are known or suspected of having MEN-2B syndrome. The 11 non-MEN-2 control subjects included 4 medical person- Improvements in cytogenetic methodology to obtain high- nel, 2 spouses of MEN patients, 2 definitely unaffected resolution karyotypes (1-3) have helped delineate two syn- MEN-2 family members, 1 subject with MEN-1 syndrome dromes that cause congenital malformations and predispose (parathyroid and pituitary tumors and suspected pancreatic the host to specific neoplasia: (i) a chromosome 13q deletion islet cell tumors), 1 subject with a pheochromocytoma asso- in the retinoblastoma-mental retardation-congenital malfor- ciated with von Hipple-Lindau disease, and her normal sib- mation complex (4-6) and (ii) a chromosome lip deletion in ling. the aniridia-Wilms tumor association (7-9). In mendelian Cytogenetic Analysis. Chromosome preparations were dominant retinoblastoma or Wilms tumor without mental re- made from amethopterin-synchronized, phytohemaggluti- tardation or other malformations, the tumor cells may have a nin-stimulated lymphocyte cultures with minor modifica- karyotype abnormality, but the peripheral blood chromo- tions of the method detailed by Yunis et al. (1). In our tech- somes of the patient appear normal (10-12). In two families nique, vinblastine sulfate (0.0625 pug/ml) replaced colchi- with autosomal dominant predisposition to renal-cell carci- cine, and the slides were G-banded by trypsin with Giemsa. noma, the affected individuals had either an inherited (13) or During the first several years of our investigations of high- a tumor-acquired (14) balanced translocation. Many other resolution karyotypes in MEN-2 (16-18), neurofibromatosis autosomal dominant conditions have a predisposition to can- (16), XX males (21), Prader Willi and William syndromes, cer (15). Most are probably due to point mutations, but the familial breast cancer, and other disorders (unpublished findings in retinoblastoma, Wilms tumor, and renal-cell car- data), a protocol for investigating suspected minute chromo- cinoma suggest that some may exhibit visible chromosome some abnormalities was developed, which was used in the aberrations with high-resolution cytogenetic methodology. double-blind study. Each blood sample was delivered to the In 1981 we suggested that individuals with mendelian dom- cytogenetics laboratory identified by a code number known inant multiple endocrine neoplasia type 2A syndrome carry only to the clinical geneticist. Several high-resolution GTG- an interstitial 20p deletion (16-18). However, Hsu et al. (19) banded slides were prepared, and the location was noted of and Emmertsen et al. (20) were not able to distinguish the all prometaphase and late prophase spreads (approximately deletion in affected members of one MEN-2 family studied 850 and 1,000 bands per haploid set, respectively). Two or by each group. We describe here our findings in a double- three cytogeneticists independently scored each chromo- blind study of MEN-2 patients and controls, scoring high- some 20 pair from these cells for the 20p deletion. All scoring resolution G-banded cells for the presence or absence of the was done through the microscope to assure optimal resolu- 20p deletion. tion. The majority of cells located were not considered scor- able because one or both 20p arms overlapped with other MATERIALS AND METHODS chromosomes, the critical band 20pl2 was bent or twisted or Study Subjects. The blind study included nine indepen- had chromatids overlapped, or the chromosome 20 homo- dently scored blood samples from eight affected members of logs clearly differed in their degree of condensation. After four MEN-2A families, eight independently scored blood both (or all three) cytogeneticists concurred that a subject samples from six affected members of five MEN-2B fam- had the deletion or not, the 'code' was broken. Agreement ilies, and 13 independently scored blood samples from 11 among the cytogeneticists has been attained in every case for The Abbreviation: MEN, multiple endocrine neoplasia. publication costs of this article were defrayed in part by page charge *Present address: Arkansas Children's Hospital, 804 Wolfe Street, payment. This article must therefore be hereby marked "advertisement" Little Rock, AR 72202. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 2525 Downloaded by guest on September 26, 2021 2526 Medical Sciences: Babu et al. Proc. Natl. Acad Sci. USA 81 (1984) which a sufficient number (at least 9-20) of adequate chro- MEN-2 patients carry a 20p deletion and all non-MEN-2 sub- mosome 20 pairs was available for scoring. jects have normal chromosomes 20, there were 25 correct and 5 incorrect assignments to phenotype based on the RESULTS karyotype scores (X2 = 13.25; P << 0.001). Likewise, 20 of The double-blind study included 30 independently scored 25 blood samples from different individuals (subsequent blood samples from 25 subjects (Table 1). Nine samples from samples excluded) were identified correctly in the double eight non-MEN-2 control subjects were scored as having blind study (X2 = 9.00; P < 0.001). normal chromosomes 20. Two control samples were scored A nonblind reassessment was carried out on the samples as having a 20p deletion (karyotype 20p-), clearly in error. that had scores inconsistent with the phenotype of the sub- Unbeknownst to the cytogeneticists, a second blood sample ject. The two new mutant MEN-2B patients scored as having from each of these two individuals was added to the double- normal chromosomes 20 (Table 1, ALS II-1 and MAR II-4) blind study, and both samples were scored as having normal still appeared to have normal chromosomes 20 on indepen- chromosomes 20. Nine blood samples from eight MEN-2A dent reevaluation by all three cytogeneticists. The other patients were scored as having the 20p deletion (Fig. 1). three inconsistent results (Table 1, MEN-2B patient TRU Three samples from MEN-2B patients were scored as having III-1 and controls LIGOR and EST II-3) had been scored the 20p deletion, and three were scored as having normal concurrently, and the errors in scoring were discovered in chromosomes 20. One sample that scored as normal and one the same code-breaking ceremony. It is our subjective inter- that scored as karyotype 20p- were from siblings in the TRU pretation that these errors occurred because we had inadver- kindred. Unbeknownst to the cytogeneticists, a second tently relaxed our scoring criteria for these samples by at- blood sample from these two patients was added to the dou- tempting to analyze too many cells with substandard G- ble-blind study, and both samples were scored as having the banding and by being insufficiently attuned to the variability 20p deletion. The other two MEN-2B patients whose blood between homologs caused by differential chromosome con- samples were scored as having normal chromosomes 20 ap- densation. pear by pedigree analysis to represent new mutants. samples from 11 DISCUSSION In summary, 11 of 13 non-MEN-2 control Based on our double-blind study, we interpret the chromo- subjects were scored as having normal chromosomes 20, and dominant MEN-2A and 14 of 17 blood samples from 14 MEN-2 patients were scored some abnormality in the autosomal If one assumes that all MEN-2B syndromes as an interstitial deletion involving part as having the 20p deletion (Table 2). of the light-staining subband 20pl2.2 and designate the de- Table 1. Findings in the double-blind chromosome study of the fect as 46,XX or XY,del(20)(p12.2p12.2). Three less likely chromosome 20 deletion in MEN-2A and MEN-2B syndromes interpretations of our observation are that the 20p deletion is a normal chromosome variant, part of a chromosome 20 in- Subject Karyotype Phenotype version, or part of an insertional translocation.